WO2021167175A1 - Pharmaceutical composition for preventing or treating cancer, comprising mtor-signaling inhibitor as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an mTOR-signaling inhibitor as an active ingredient. The composition of the present invention exhibits the efficacy of inhibiting the growth of cancer cells and killing cancer cells, and thus can be used as an anticancer agent.

Description

Pharmaceutical composition for preventing or treating cancer comprising an emtor signaling inhibitor as an active ingredient

This patent application claims priority to Korean Patent Application No. 10-2020-0021549 filed with the Korean Intellectual Property Office on February 21, 2020, the disclosure of which is incorporated herein by reference.

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

Cancer is the most common and serious disease that threatens human health, and research and development of anticancer drugs is important for prolonging the lifespan of cancer patients. In recent years, cancer treatment methods have made a lot of progress due to the rapid development of oncogenomics and molecular pharmacology and the development of new anticancer drugs, but there is still a need to develop new therapeutic agents. Colorectal cancer is a malignant tumor that occurs in the appendix, colon, and rectum, and occurs in the mucous membrane, the innermost surface of the large intestine. Colorectal cancer is the second most common cancer in Korea after gastric cancer, and its incidence has increased sharply with the westernization of eating habits. Its rate of increase continues to increase.

Treatment of colorectal cancer can be divided into surgical resection, chemotherapy, and radiation therapy. Polyps, which are the pre-stage of colorectal cancer, or early colon/rectal cancer limited to polyps, can be treated with polypectomy. In the treatment of colorectal cancer, since Fluorouracil (5-FU) developed in the 1970s, five anticancer drugs such as irinotecan, oxaliplatin, capecitabine, TAS-102, and epithelial cell growth cetuximab, Panitu, a targeted anticancer agent that targets epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), or vascular endothelial growth factor receptor (VEGF-R) Five targeted anticancer drugs, including panitumumab, bevacizumab, aflibercept, and regorafenib, have been approved by the US FDA and are being used. However, there is still an urgent need to develop a treatment for colorectal cancer with excellent anticancer effect, stability, and absorption into the body.

mTOR (mechanistic target of rapamycin) protein is a serine/threonine kinase and is a key signaling factor that regulates cell growth, cell cycle, protein synthesis and glucose metabolism. In particular, as a key protein for regulating cell growth signaling, its activity is abnormally increased in 30% of solid cancer cells, and it is known that these emtors and higher-level factors (PI3K, Akt) are the most changed in cancer cells. Activation of emtor signals in cancer is caused by mutations in the emtor gene itself, high-level oncogenes (PI3K, Akt) that enhance the activity of emtor, and mutations in tumor suppressor factors (eg PTEN, TSC1/2). . Therefore, inhibition of emtor-associated signaling may induce cell autophagy along with protein synthesis inhibition, lipid synthesis inhibition, and cell growth inhibition, leading to cell death. Therefore, there is a need for the development of an emtor-related signaling inhibitor for the inhibition of cancer cell growth and cancer cell death.

The present inventors made intensive research efforts to develop compounds for inhibiting cancer cell growth and killing cancer cells. As a result, the present invention was completed by identifying that the composition comprising an mTOR signaling inhibitor as an active ingredient is effective in treating cancer.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

The emtor signal transduction inhibitor refers to a substance that inhibits the activity of emtor itself or inhibits the activity of an upper-level factor that increases the activity of emtor. The target whose activity is inhibited by the emtor signaling inhibitor may include, but is not limited to, emtor, PI3K, Akt, S6K, and S6.

In one embodiment of the present invention, the emtor signaling inhibitor is lomitapide.

Lomitapide (trade names Jaxtapid (US), Lojuxta (EU)) is a rare disease treatment developed by Aegerion Pharmaceuticals and is used as a cholesterol lowering agent in the treatment of familial hypercholesterolemia. On December 21, 2012, the U.S. Food and Drug Administration (FDA) reported that LDL cholesterol, total cholesterol, apolipoprotein B, and non-high-density lipoprotein Lomitapide was approved as a drug to reduce high-density HDL) cholesterol.

The IUPAC name of lomitapide is N- (2,2,2-trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[1,1'-biphenyl] ] 2-yl] carbonyl] amino] -1-piperidinyl] butyl] -9 H - fluoren-9-carboxylic a copy polyimide

In one embodiment of the present invention, the lomitapide is represented by the following formula (I).

[Formula I]

In one embodiment of the present invention, the emtor signaling inhibitor is lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

In one embodiment of the present invention, the cancer is a solid cancer.

As used herein, the term "solid cancer" has characteristics distinguishing it from blood cancer, and includes bladder, breast, intestine, kidney, lung, liver, brain, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate and skin. It is a cancer consisting of a mass caused by abnormal cell growth in various solid organs such as

In one embodiment of the present invention, the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer , nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, Pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external reproductive system It is selected from the group consisting of stage cancer, female urethral cancer, bone tumor, duodenal cancer, fibrosarcoma, and skin cancer, but is not limited thereto.

In one embodiment of the present invention, the cancer is a hematologic cancer.

As used herein, the term “hematologic cancer” refers to cancer occurring in components constituting blood, and refers to malignant tumors occurring in blood, hematopoietic organs, lymph nodes, lymphatic organs, and the like.

In one embodiment of the present invention, the hematologic cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. is selected from, but not limited thereto.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration , intracerebral administration, intracranial administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe an effective dosage (a pharmaceutically effective amount) for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.

As used herein, the term “prevention” refers to the prevention or protective treatment of a disease or disease state. As used herein, the term “treatment” refers to reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. At this time, the dosage form may be prepared in various ways such as oral medicine, injection, etc., in the form of a solution, suspension or emulsion in oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, dispersant or stable Additional topics may be included.

According to an embodiment of the present invention, the pharmaceutical composition comprising lomitapide of the present invention has the effect of inhibiting the emtor signaling process and increasing the expression of proteins involved in the autophagy mechanism of cells.

As used herein, the term “autophagy” is a destructive process that naturally decomposes unnecessary or non-functional cellular components in the control process of cells.

The autophagy-related genes include ULK1, mTOR, ATG family, DDIT4, and the like.

ULK1 is a gene encoding a protein kinase required for the initiation of autophagy, and ULK1 activity is suppressed under conditions of high mTOR protein activity. When mTOR activity is inhibited, the increase in ULK1 activity directs it to initiate autophagy-related signaling.

As a protein kinase, mTOR is a key factor in the inhibition of autophagy as well as the regulation of cell growth and activity. mTOR directly phosphorylates ULK1 and various autophagy proteins to mediate the inhibition of autophagy. Conversely, autophagy is actively promoted under conditions in which mTOR activity is inhibited.

ATG family genes are Autophagy-Related Genes composed of more than 30 key genes involved in the initiation and progression of autophagy. These genes are necessary for the whole process of autophagy, including the initiation of autophagy, the formation of the autophagic membrane, the recognition of the substrate to be degraded, and the fusion reaction between the autophagy membrane and lysosome.

DDIT4, also known as REDD1, is a protein that inhibits mTOR through the TSC protein. Therefore, an increase in DDIT4 is a gene that increases the activity of autophagy by inducing inhibition of mTOR.

In addition, according to an embodiment of the present invention, the pharmaceutical composition of the present invention has the effect of inhibiting the growth of cancer cells and the effect of inhibiting the growth of tumors in vivo. Therefore, the pharmaceutical composition of the present invention is cancer, more specifically blood cancer, skin cancer, colorectal cancer, brain cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, duodenal cancer, fibrosarcoma, kidney cancer, liver cancer, lung cancer, pancreatic cancer, It has anticancer effects against prostate cancer, stomach cancer, bone tumor, endometrial cancer, etc.

According to another aspect of the present invention, the present invention provides a method for preventing or preventing cancer comprising administering to a subject a pharmaceutical composition comprising the above-described mTOR signaling inhibitor of the present invention as an active ingredient. provide a treatment method.

As used herein, the term "administration" or "administer" refers to directly administering a therapeutically or prophylactically effective amount of a composition of the present invention to a subject (individual) suffering from, or likely to suffer from, the subject disease. It means that the same amount is formed in the body of

The "therapeutically effective amount" of the composition means an amount of the composition sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is administered, and includes a "prophylactically effective amount".

In addition, as used herein, the term "subject (subject)" is a mammal, including humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons and rhesus monkeys. . Most specifically, the subject of the present invention is a human.

Since the method for preventing or treating cancer of the present invention includes administering the pharmaceutical composition according to an aspect of the present invention, the description thereof is omitted to avoid excessive redundancy of the present specification for overlapping content. .

The features and advantages of the present invention are summarized as follows:

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

The composition of the present invention can be used as an anticancer agent by inhibiting the growth of cancer cells and exhibiting the efficacy of killing cancer cells.

1 shows the chemical formula of lomitapide.

Figure 2 shows the results of analyzing the shape of the cells after treating the normal human intestinal epithelial cells and colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.

Figure 3 shows the results of analyzing the cell viability after treating the normal human intestinal epithelial cells and colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.

4 shows the results of analysis of cell viability after treatment with DMSO control drug and lomitapide for three representative colorectal cancer cell lines (HCT116, HT29, SW480).

Figure 5a shows the results of analyzing the cell viability after lomitapide treatment for brain cancer cell lines.

Figure 5b shows the results of analyzing the viability of cells after treatment with lomitapide targeting breast cancer cell lines.

Figure 5c shows the results of analyzing the viability of the cells after lomitapide treatment on a blood cancer cell line.

Figure 5d shows the results of analyzing the cell viability after treatment with lomitapide targeting colon cancer cell lines.

Figure 5e shows the results of analyzing the viability of cells after treatment with lomitapide targeting lung cancer cell lines.

Figure 5f shows the results of analyzing the viability of the ovarian cancer cell line after treatment with lomitapide.

5g shows the results of analyzing the cell viability of gastric cancer cell lines after treatment with lomitapide.

Figure 5h shows the results of analyzing the cell viability after treatment with lomitapide in bladder cancer, bone cancer, duodenal cancer, endometrial cancer, fibrosarcoma, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, and skin cancer cell lines.

6 shows the results of analyzing proteins related to emtor signaling in cells after treatment with a DMSO control drug and lomitapide for colon cancer cells HCT116 through Western blot.

Figure 7a shows the results of analysis of proteins related to emtor signaling and autophagy in the colon cancer cells SW480 after treatment with DMSO control drug and lomitapide through Western blot.

Figure 7b shows the results of analysis of proteins related to emtor signaling and autophagy in cells after treatment with DMSO control drug and lomitapide for colorectal cancer cells HT29 through western blot.

Figure 7c shows the results of analyzing the proteins related to emtor signaling and autophagy in the cells after treatment with DMSO control drug and lomitapide for breast cancer cells MDA-MB-231 through Western blot.

Figure 7d shows the results of analyzing the proteins related to emtor signaling and autophagy in the cells after treatment with DMSO control drug and lomitapide for breast cancer cells MDA-MB-468 through Western blot.

8 shows the results of analyzing the protein related to autophagy of cells after treatment with DMSO control drug and lomitapide for colon cancer cells HCT116 through Western blot.

Figure 9 shows the results of analysis of apoptosis by autophagy after treating the human colorectal cancer cell line HCT116 with DMSO control drug, lomitapide, and 3-methylamine (3-MA), respectively, through Western blot.

10 shows the results of analysis of apoptosis by autophagy after the human colon cancer cell line HCT116 was treated with a DMSO control drug, lomitapide, and 3-methylamine (3-MA), respectively, through a microscope.

11 shows the results of analyzing the significant gene-mechanism relationship using RNA-seq analysis after treating the human colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.

12 shows the results of analyzing significant genes related to the autophagy mechanism using RNA-seq analysis after treating the human colon cancer cell line HCT116 with a DMSO control drug and lomitapide, respectively.

13 shows the results of measuring the degree of cancer cell colony proliferation after treating colon cancer cells HCT116 with DMSO control drug and lomitapide.

Figure 14a shows the results of comparing and analyzing the change in tumor size with the control group after treatment with lomitapide (50 mg/Kg) of colorectal cancer cells HCT116 xenografted into mice.

Figure 14b shows the results of analyzing the change in tumor size after treatment with lomitapide (25, 50 mg/Kg) of colorectal cancer cells HCT116 xenografted into mice.

Figure 14c shows the results of analyzing the change in tumor size after treatment with lomitapide (10 mg/Kg) of colon cancer cells xenografted to mice HCT116.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1: Cell culture method

NCM460 cells (human colon normal cells), HCT116 cells (human colon cancer cells, p53 wild-type), HT29 cells (human colon cancer cells, p53 mutant), SW480 cells (human colon cancer cells, p53 mutant), MDA-MB-231 cells (human breast cancer cells) and MDA-MB-468 cells (human breast cancer cells) were purchased from ATCC (American Type Culture Collection, Virginia, USA) and used.

NCM460, HCT116 cells were grown in McCoy's 5a medium (Sigma Aldrich, Missouri, USA) supplemented with 2 mM glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). 37 ℃, 5% CO ₂ Incubated under conditions. HT29, SW480 cells were cultured in RPMI medium supplemented with 2 mM glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Sigma Aldrich, Missouri, USA). 37 ℃, 5% CO ₂ Incubated under conditions. MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM medium (Sigma Aldrich, Missouri, USA) supplemented with 2 mM glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C, 5 % CO ₂ Incubated under conditions.

Example 2: Viability assay of cells treated with lomitapide

Example 2-1. Viability analysis of lomitapide-treated colon cancer cells

NCM460, HCT116, HT29, SW480 cells were seeded in 96-well plates at ^{a density of 10 4} cells/well, incubated at 37 °C for 24 hours, and then at various concentrations (0, 1, 2, 5, 10 μM). It was treated with tapide (Sigma Aldrich, Missouri, USA). After lomitapide treatment Plates were incubated at 5% CO ₂ , 37° C. for 24 hours. Thereafter, 100 μL of assay reagent (CellTiter-Glo® Reagent) was added to each well and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA). %) was calculated. The results are shown in FIGS. 2 to 4 .

Through the above experiment, it was confirmed that in the case of the anticancer cells treated with lomitapide, the survival rate of the cells was decreased compared to that of the normal cells ( FIG. 2 ). The above results indicate that lomitapide has a cancer cell-specific anticancer effect.

In addition, through the above experiment, it was confirmed that the cell viability decreased as the dose of lomitapide increased in three representative colorectal cancer cell lines (HCT116, HT29, SW480) ( FIGS. 3 and 4 ).

The above results indicate that lomitapide is very effective in killing various colorectal cancer cells.

Example 2-2. Viability analysis of lomitapide-treated cancer cells

Cell viability was analyzed by treatment with various concentrations of lomitapide for 120 cancer cells other than colon cancer cells.

To analyze the viability of cells, cells were treated with lomitapide _{, incubated for 72 hours at 37 °C in 5% CO 2} or 10% CO ₂ conditions, equilibrated to room temperature for 1 hour, and CellTiterGlo reagent (Promega) was added. After 1 hour, the luminescence was measured, and the cell viability (%) was calculated therefrom. 120 types of cells were purchased from ATCC (American Type Culture Collection, Virginia, USA) and used.

The results of analyzing cell viability for various cancer cells are as follows (Table 1, FIGS. 5A to 5H).

division	cell name	IC50 (μM)	target organ
One	A172	1.90	brain
2	A2058	1.40	skin
3	A2780	2.70	ovary
4	A375	1.80	skin
5	A498	2.90	kidney
6	A549	3.60	lung
7	AsPC-1	3.60	pancreas
8	BEN	4.20	lung
9	BT-20	5.00	breast
10	BxPC-3	5.70	pancreas
11	Caki-1	3.60	kidney
12	Caki-2	2.80	kidney
13	Colo 205	3.40	colon
14	COR-L279	2.70	lung
15	COV434	3.10	ovary
16	DLD-1	4.50	colon
17	DU-145	5.00	prostate
18	DV90	5.20	lung
19	EFM-19	3.60	breast
20	EFM-192A	4.00	breast
21	EFO-27	4.00	ovary
22	EPLC-272H	3.10	lung
23	H1299	4.80	lung
24	H2228	5.30	lung
25	H4	4.60	brain
26	H460	3.80	lung
27	HCC 1569	2.90	breast
28	HCC38	6.00	breast
29	HCC827	4.10	lung
30	HCT116	5.00	colon
31	HCT-15	3.50	colon
32	HEC-1-A	4.90	endometrium
33	HEC-1-B	6.90	endometrium
34	Hep3B2.1-7	2.70	liver
35	HL-60	3.30	blood
36	Hs 746T	2.50	stomach
37	HT-1080	3.90	fibrosarcoma
38	HT-29	1.60	colon
39	Hutu 80	3.00	duodenum
40	Ishikawa	3.60	endometrium
41	J82	3.60	bladder
42	JIMT-1	3.30	breast
43	Jurkat	3.00	blood
44	JVM-3	4.00	blood
45	K562	2.10	blood
46	KARPAS 299	1.40	blood
47	Kato III	5.40	stomach
48	KG-1	5.70	blood
49	KMS-12-BM	3.50	blood
50	LN229	2.70	brain
51	LnCap	3.50	prostate
52	LOU-NH91	5.70	lung
53	LOVO	3.40	colon
54	LP-1	5.40	blood
55	M07e	4.20	blood
56	MCAS	4.50	ovary
57	MCF-7	2.80	breast
58	MDA MB 231	3.20	breast
59	MDA MB 435	3.00	skin
60	MEC-1	3.30	blood
61	Mia PaCA 2	2.90	pancreas
62	MKN-1	3.70	stomach
63	MKN-45	3.90	stomach
64	MOLM-13	6.30	blood
65	MOLT-4	3.60	blood
66	MV4-11	6.20	blood
67	NCI-H1048	5.50	lung
68	NCI-H1437	3.10	lung
69	NCI-H1563	5.30	lung
70	NCI-H1573	4.70	lung
71	NCI-H1581	3.70	lung
72	NCI-H1703	5.70	lung
73	NCI-H1838	4.00	lung
74	NCI-H2009	3.30	lung
75	NCI-H2110	3.30	lung
76	NCI-H2286	3.40	lung
77	NCI-H292	3.00	lung
78	NCI-H441	3.20	lung
79	NCI-H82	3.20	lung
80	NCI-H838	3.80	lung
81	NCI-N87	3.30	stomach
82	OCI-AML3	2.10	blood
83	OCI-AML5	2.70	blood
84	OCI-LY19	2.80	blood
85	OPM-2	4.40	blood
86	OV56	4.80	ovary
87	OVCAR-3	5.30	ovary
88	OVK18	3.30	ovary
89	P31/FUJ	3.10	blood
90	PANC-1	4.50	pancreas
91	PC3	4.30	prostate
92	RERF-LC-Ad2	2.60	lung
93	RERF-LC-MS	3.20	lung
94	RKO	3.30	colon
95	RL95-2	3.20	endometrium
96	RPMI 8226	2.10	blood
97	Saos-2 EC	3.60	bone
98	SCLC-21H	3.00	lung
99	SJSA-1	3.10	bone
100	SK.BR-3	3.90	breast
101	SK-ES-1	3.50	bone
102	SK-LU-1	4.60	lung
103	SK-MEL-3	1.60	skin
104	SK-N-FI	3.40	brain
105	SK-N-MC	2.40	brain
106	SK-N-SH	3.20	brain
107	SK-OV3	3.30	ovary
108	SNU-1	2.80	stomach
109	SNU-216	3.10	stomach
110	SNU840	2.30	ovary
111	SW480	2.50	colon
112	SW620	3.60	colon
113	SW948	4.50	colon
114	T84	3.20	colon
115	T98G	3.80	brain
116	U118MG	2.50	brain
117	U251MG	5.40	brain
118	U2OS	2.80	bone
119	U87MG	3.10	brain
120	U-937	2.30	blood

Through the above experiment, it was confirmed that lomitapide had anticancer effects at very low concentrations in various carcinomas and cancer cell lines, and the types were blood cancer, skin cancer, colorectal cancer, brain cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, duodenal cancer, fibrosarcoma, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gastric cancer, bone tumor, endometrial cancer, and the like.

Example 3: Cell signaling assay of lomitapide-treated cells

In order to confirm the function of lomitapide in HCT116, SW480, HT29, MDA-MB-231, and MDA-MB-468 cell lines, the signaling effect of cells when lomitapide was added was examined.

Lomitapide-treated at various concentrations (0, 5, 10 μM) Cells were lysed with RIPA buffer containing a protease-inhibitor cocktail. Whole-cell lysate was incubated on ice for 30 minutes, then centrifuged at 4 °C, 13,300xg for 15 minutes, and the supernatant was collected. As a control, 1 μM of Torin, known as an mTOR inhibitory component, was treated. HCT116 cells were used.

For Western blot analysis, the supernatant obtained above was separated by loading on a 10% SDS-PAGE gel, and the separated protein was blotted on a PVDF membrane. Then, the antibody was added to the blot as follows (Table 2), and incubated at 4° C. for 12 hours.

cell	antibody
HCT116	anti-p-AKT, anti-p-mTOR, anti-p-S6K, anti-p-S6, anti-p-ERK, anti-AKT, anti-S6K, anti-S6 (Cell Signaling Technology, Massachusetts, USA) , anti-alpha-tublin antibody (Sigma Aldrich, Missouri, USA)
SW480	Anti-p-S6K, anti-p-S6, anti-S6K, anti-S6, anti-LC3 (Cell Signaling Technology), anti-alpha-tublin antibody (Sigma Aldrich)
HT29
MDA-MB-231
MDA-MB-468

Then, the blot was washed with TBST (a mixture of tris-buffered saline (TBS) and Tween 20), and at 37 °C, 1 with horseradish peroxidase-conjugated secondary antibody (Cell signaling). for time After incubation and washing, enhanced chemiluminescence (ECL; Amersham) was detected.

Through the above experiment, it was confirmed that lomitapide significantly reduced phosphorylation levels of target proteins (p-AKT, p-mTOR, p-S6K, p-S6) in a concentration-dependent manner in HCT116 ( FIG. 6 ). In addition, it was confirmed that lomitapide significantly reduced phosphorylation levels of target proteins (p-S6K, p-S6) in SW480, HT29, MDA-MB-231, and MDA-MB-468. It was confirmed that the induction of LC3I to LC3II was increased ( FIGS. 7A to 7D ). These results indicate that lomitapide is very effective in inhibiting emtor (mTOR) and the upper signaling pathways of emtor (AKT, p-S6K, p-S6).

Example 4: Autophagy assay of cells treated with lomitapide

In order to confirm the correlation between the cellular autophagy function of lomitapide in the HCT116 cell line, the signaling effect of cells when lomitapide was added was examined.

Lomitapide-treated at various concentrations (0, 5, 10 μM) for analysis of changes in cell signaling involved in cell autophagy HCT116 cells were analyzed by Western blot. For Western blotting, the supernatant obtained in Example 3 was loaded on a 10% SDS-PAGE gel to separate, and the separated protein was blotted on a PVDF membrane. Then, anti-p-AMPK, anti-LC3, and anti-p-ULK1 antibodies (Cell Signaling Technology, Massachusetts, USA) were added to the blot, and incubated at 4° C. for 12 hours. Then, the blot was washed with TBST (a mixture of tris-buffered saline (TBS) and Tween 20), and at 37 °C, 1 with horseradish peroxidase-conjugated secondary antibody (Cell signaling). for time After incubation and washing, enhanced chemiluminescence (ECL; Amersham) was detected.

Through the above experiment, it was confirmed that lomitapide significantly increased the phosphorylation level of AMPK, decreased the phosphorylation level of ULK1, and significantly increased the induction of LC3I to LC3II in a concentration-dependent manner ( FIG. 8 ). These results indicate that lomitapide has the effect of greatly increasing the expression of proteins involved in the autophagy mechanism.

In addition, in the case of simultaneous treatment of lomitapide and 3-methylamine (3-MA, Sigma Aldrich, Missouri, USA), which is an autophagy function inhibitor, in order to confirm the correlation between the cellular autophagy function of lomitapide in the HCT116 cell line. The signaling effect of the cells and the viability of the cells were examined.

Lomitapide-treated at 5 μM for analysis of changes in cell signaling involved in cell autophagy HCT116 cells and HCT116 cells treated with 1-mM 3-MA and 5 μM of lomitapide were analyzed by Western blot. The Western blotting procedure is the same as the Western blotting procedure described above except that anti-LC3 was used as the antibody.

In addition, for cell viability analysis, HCT116 cells were inoculated into a 96-well plate at ^{a density of 10 4} cells/well, and incubated at 37°C for 24 hours, 5 μM of lomitapide, 3-MA 1 mM or 5 It was treated with μM lomitapide and 1-mM 3-MA. after processing Plates were incubated at 5% CO ₂ , 37° C. for 24 hours. Thereafter, 100 μL of an assay reagent (CellTiter-Glo® Reagent) was added to each well and the results were observed under a microscope.

Through the above experiment, it was confirmed that HCT116 cells treated with lomitapide and 3-MA significantly reduced the induction of LC3I to LC3II ( FIG. 9 ). In addition, it was confirmed that cell death was inhibited in the case of HCT116 cells treated with lomitapide and 3-MA through cell viability analysis (FIG. 10).

The above results indicate that lomitapide of the present invention inhibits AMPK and mTOR signaling processes, thereby increasing the expression of cellular autophagy mechanism proteins.

Example 5: Analysis of significant gene expression and mechanism of lomitapide-treated cells (RNA-seq assay)

Gene set enrichment analysis (GSEA) and Pathway enrichment analysis were performed to analyze genes that are significantly expressed through RNA-seq experiments and related mechanisms to confirm the relationship between the drug mechanism of lomitapide and genes. was carried out through

GSEA is an analysis method for extracting a significant gene-set in which expression values of two classes show a statistically significant difference from among various gene-sets constructed based on biological characteristics.

In order to finally detect significant gene-sets, genes with specific functions among all genes used in RNA-seq experiments using a gene annotation database (KEGG pathway, Gene Ontology, etc.) with various biological information about genes were identified. Various gene-sets were discovered by grouping, and significant genes were determined by referring to the difference in expression values between two classes within each gene-set, and statistically significant gene-sets were finally detected based on this.

As for the mechanisms to which a significant gene set belongs, cancer-related mechanisms related to the Emtor mechanism including autophagy were detected, and in addition, immune, aging, and diabetes-related mechanisms were detected ( FIG. 11 , Table 3).

division	gene function	GeneRatio	associated disease
One	MAPK signaling pathway	0.31	cancer
2	Kaposi sarcoma-associated herpesvirus infection	0.4	cancer
3	Viral carcinogenesis	0.42	cancer
4	breast cancer	0.37	breast cancer
5	FoxO signaling pathway	0.52	cancer
6	Apoptosis	0.35	cancer
7	TNF signaling pathway	0.62	Inflammation
8	osteoclast differentiation	0.81	osteoporosis
9	IL-17 signaling pathway	0.45	inflammation, cancer
10	colorectal cancer	0.58	colorectal cancer
11	Transcriptional misregulation in cancer	0.4	cancer
12	Hepatitis B	0.33	inflammation, cancer
13	Non-alcoholic fatty liver disease (NAFLD)	0.33	Nonalcoholic hepatitis, cirrhosis and liver failure
14	Parathyroid hormone synthesis, secretion and action	0.53	Calcium metabolism, cancer
15	Adrenergic signaling in cardiomyocytes	0.62	heart disease
16	Epithelial cell signaling in Helicobacter pylori infection	0.38	gastritis, stomach cancer
17	Human T-cell leukemia virus 1 infection	0.35	leukemia
18	Cytokine-cytokine receptor interaction	0.37	inflammation, cancer
19	Autophagy - animal	0.6	Cancer, metabolic disease
20	C-type lectin receptor signaling pathway	0.16	immunity, cancer
21	Spliceosome	0.88	-
22	Relaxin signaling pathway	0.6	heart disease
23	AGE-RAGE signaling pathway in diabetic complications	0.53	diabetic complications
24	Pertussis	0.33	whooping cough
25	PI3K-Akt signaling pathway	0.24	cancer
26	Fluid shear stress and atherosclerosis	0.31	arteriosclerosis
27	ErbB signaling pathway	0.69	cancer
28	NF-kappa B signaling pathway	0.46	inflammation, cancer
29	Pathways in cancer	0.3	cancer
30	Neuroactive ligand-receptor interaction	0.52	-
31	Endocrine resistance	0.41	metabolic disease
32	Signaling pathways regulating pluripotency of stem cells	0.39	cancer
33	Peroxisome	0.83	-
34	Protein processing in endoplasmic reticulum	0.26	-
35	Shigellosis	0.38	bacterial dysentery

In addition, it was found that autophagy-related genes, such as ATG family, ULK1, and DDIT4, were significantly expressed through Differentially Expressed Genes analysis (DEG analysis) related to the autophagy mechanism (FIG. 12). DEG analysis is an analysis that selects a candidate group for a significant gene (Differentially Expressed Genes) with a difference in expression between the control group and the control group by measuring and statistically processing the expression value of the gene.

As can be seen from the above results, the effect of lomitapide on cancer cells is shown through i) inhibition of the emtor signaling mechanism and ii) gene expression related to autophagy.

Example 6: Cell colony forming assay of lomitapide-treated cells

In order to confirm the correlation between lomitapide and cancer cell colony growth rate in HCT116 cell line, the cancer cell colony growth rate was examined when lomitapide was added to the wells culturing HCT116 cells.

HCT116 cells were seeded in a 12-well plate at ^{a density of 10 5} cells/well, and after incubation at 37° C. for 24 hours, the cells were treated with 0, 5 μM lomitapide. After lomitapide treatment Plates were incubated at 5% CO ₂ , 37° C. for 48 hours. Thereafter, 500 μL of crystal violet was added to each well, and the cells were stained at room temperature for 10 minutes to analyze the proliferation of the cells.

Through the experiment, it was confirmed that the cell proliferation rate in the wells treated with lomitapide was significantly lower than that in the wells not treated with lomitapide ( FIG. 13 ).

These results indicate that lomitapide has a very excellent anticancer effect.

Example 7: Analysis of the anticancer effect of lomitapide confirmed through animal experiments

In order to confirm the anticancer effect of lomitapide in the mouse xenograft model, the size of the tumor was examined after lomitapide was treated in the tumor-transplanted mice.

Animal experiments were performed according to guidelines approved by the Animal Experimental Ethics Committee of the Korea Advanced Institute of Science and Technology. HCT116 cells (4x10 ⁶ ) were implanted by subcutaneous injection into male BALB/c nude mice aged 8-12 weeks. After the mean tumor volume ^{reached 50 mm 3} , mice were randomly assigned to two different groups (5 mice/group). The mouse body weight and tumor diameter were measured once every two days. Tumor volume was evaluated according to the general formula 0.5x(width) ² x(Length) using a caliper, and Student's T test was used to determine P-values. For lomitapide treatment, 1, 2, 3, 7, 8, 9, 13, 14, 15, and 16 days after the start of the experiment, 50 mg/kg of lomitapide was administered to the mouse. Injected into the abdominal cavity. As a control, DMSO was injected intraperitoneally into mice ( FIG. 14A ).

In addition, additional animal experiments were performed, and 25, 50 mg/kg of lomitapide was injected into the abdominal cavity of mice over a total of 5 times at intervals of 2 days after the start of the experiment. As a control, DMSO was injected intraperitoneally into mice ( FIG. 14b ).

In addition, additional animal experiments were performed, and 10 mg/kg of lomitapide was intratumoral injection of 10 mg/kg of lomitapide for a total of 5 times at an interval of 2 days after the start of the experiment. As a control, DMSO was injected intratumorally in mice ( FIG. 14c ).

Through the experiment, it was confirmed that the growth of the tumor in the lomitapide-treated mouse xenograft model was inhibited when compared to the control group (FIG. 14).

These results indicate that lomitapide has a very excellent anticancer effect.

In conclusion, the experiments described in the above Examples show that lomitapide produces anticancer effects by inhibiting emtor-related signal transduction and activating the autophagy mechanism of cells.

Claims (7)

1. A pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.
2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the emtor signaling inhibitor is lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
3. The pharmaceutical composition for preventing or treating cancer according to claim 2, wherein the lomitapide is represented by the following formula (I):

   [Formula I]

   
4. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a solid cancer.
5. The method of claim 4, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer, nasopharyngeal cancer , salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer Cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, external genital cancer, female A pharmaceutical composition for preventing or treating cancer, which is selected from the group consisting of urethral cancer, bone tumor, duodenal cancer, fibrosarcoma, and skin cancer.
6. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a blood cancer.
7. 7. The method of claim 6, wherein the hematologic cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma. The pharmaceutical composition for the prevention or treatment of cancer.

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